RESUMEN
Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers. To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors.
Asunto(s)
Apoptosis/efectos de los fármacos , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/fisiología , Interacciones Huésped-Patógeno , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Animales , Bovinos , Línea Celular , Herpesvirus Bovino 1/genética , Mutagénesis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Virales/genética , Proteína Letal Asociada a bcl/metabolismoRESUMEN
The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.
Asunto(s)
Herpesvirus Bovino 1/enzimología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Quinasa de la Caseína II/metabolismo , Bovinos , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Inmunoprecipitación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Alineación de SecuenciaRESUMEN
We characterized the expression kinetics of the transcript and protein generated from the bovine herpesvirus 1 (BHV1) homologue of the herpes simplex virus 1 (HSV1) UL12 gene that encodes a viral alkaline nuclease. The BHV1 UL12 coding sequence, which was previously shown to express in E. coli a protein exhibiting nuclease activity, is located at positions 82776-->84239 of the viral genome. Northern blot analysis of RNA from BHV1-infected cells with a single strand RNA probe complementary to UL12 detected four specific 3' coterminal viral transcripts of 4.2, 3.7, 2.2, and 0.7 kb that accumulated simultaneously from 6 to 24 hours post-infection (p.i.). S1 nuclease mapping of the UL12 capping site at position 82384 of the genome as well as the identification of a consensus polyadenylation signal at 84490-84495 allowed us to establish that the 2.2 kb transcript corresponds to that of UL 12. A UL 12 specific antiserum generated against a T7-Tag/UL12 fusion protein expressed in E. coli detected a 53 kDa protein in cell lysates from BHV1-infected cells, whose size correlated with that predicted (51,844 Da), which accumulated from 12 to 30 h p.i. Differences observed between the transcriptional and translational expression profiles suggest that the UL12 of BHV1 is regulated at both the translational and posttranslational levels. Surprisingly, the protein expression was strictly dependent on viral DNA synthesis, unambiguously demonstrating that BHV1 UL12 belongs to viral genes of the gamma2 class. This is in contrast to the HSV1 and pseudorabies homologues that are classified as early (beta) genes. Further studies will be required to determine whether these kinetic differences have any functional implications.
Asunto(s)
Herpesvirus Bovino 1/genética , Ribonucleasas/genética , Animales , Bovinos , Línea Celular , ADN/biosíntesis , Herpesvirus Bovino 1/enzimología , Cinética , Peso Molecular , ARN Mensajero/análisis , Ribonucleasas/biosíntesisRESUMEN
We have identified the product of the US3 gene of bovine herpes virus type 1 (BHV-1), which is homologous to the herpes simplex virus type 1 (HSV-1) US3 protein kinase (PK) gene. The antibodies against the BHV-1 US3 gene product reacted with a 58 kDa polypeptide in BHV-1 infected cells and the 58 kDa polypeptide purified by immuno-precipitation demonstrated PK activity. Recent reports indicating that the US3 gene of HSV-1 is involved in the blockage of apoptosis in virus infected cells. As to the apoptosis in BHV-1 infected cells, we found following: (1) no apoptosis was observed in cells infected with wild type BHV-1 and the US3 mutants (2) the apoptosis induced by the osmotic shock of sorbitol treatment was blocked when cells were infected by the wild type BHV-1 (3) the US3 mutants of BHV-1 blocked the apoptosis of sorbitol treated cell, but the suppressive effect was delayed relative to that of wild type BHV-1 (4) the other BHV-1 mutants, with the intact US3 gene but with some other non-essential gene (genes) deleted behaved similar way to the US3 mutant. It is concluded that the US3 gene of BHV-1 is not directly involved in the blockage of apoptosis in infected cells.
Asunto(s)
Herpesvirus Bovino 1/genética , Proteínas Quinasas/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Animales , Western Blotting , Bovinos , Línea Celular , Fragmentación del ADN/genética , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/crecimiento & desarrollo , Mutación , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Virales/química , Proteínas Virales/genética , Virión/químicaRESUMEN
Nucleotide sequence analysis within the unique long segment of the infectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amino acids exhibited homology to alkaline deoxyribonucleases of other herpesviruses. To determine whether this IBRV gene product has nuclease activity, the gene designated UL12 was inserted into the vector pET-28a(+) and expressed in Escherichia coli as an oligohistidine-tagged protein. Upon induction with isopropyl beta-D-thiogalactopyranoside E. coli BL21 (DE3)[pLysS] cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein. The purified protein exhibited both exonuclease and endonuclease activities, each with an alkaline pH optimum.
Asunto(s)
Desoxirribonucleasas/aislamiento & purificación , Herpesvirus Bovino 1/enzimología , Animales , Bovinos , Cromatografía de Afinidad , Clonación Molecular , Desoxirribonucleasas/biosíntesis , Desoxirribonucleasas/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Herpesvirus Bovino 1/genética , Sistemas de Lectura Abierta , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
A recombinant bovine herpesvirus type 1 (BHV-1) virus (Gal-TK) has been constructed. The Gal-TK virus contains a chimeric reporter/marker gene coding for bacterial beta-galactosidase (beta-gal gene) that was inserted stably within the viral TK gene. This resulted in inactivation of the TK gene. The beta-gal gene is under the regulation of a strong, human cytomegalovirus-immediate early (HCMV-IE) promoter and is expressed as an authentic viral-coded gene. Even though the one-step growth kinetics of the recombinant and parent viruses were similar, the recombinant virus yielded less than the parent virus on Madin-Darby bovine kidney cells. After intranasal inoculation, the engineered virus was virtually avirulent for colostrum-deprived new-born calves. Similar to the parent virus, the recombinant virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was reduced significantly. The progeny viruses recovered from nasal swabs of animals inoculated with the recombinant and the Cooper strains of BHV-1 were easily distinguishable based on the beta-gal marker.
Asunto(s)
Infecciones por Herpesviridae/fisiopatología , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/patogenicidad , Timidina Quinasa/biosíntesis , Animales , Bovinos , Línea Celular , Clonación Molecular , Citomegalovirus/genética , Genes Inmediatos-Precoces , Genes Reporteros , Herpesvirus Bovino 1/enzimología , Humanos , Riñón , Cinética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Recombinación Genética , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence down-stream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl beta-D-thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.
Asunto(s)
ADN Glicosilasas , Genes Virales , Herpesvirus Bovino 1/genética , N-Glicosil Hidrolasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Secuencia de Consenso , Nucleótidos de Desoxiuracil/metabolismo , Escherichia coli/genética , Herpesvirus Bovino 1/enzimología , Peso Molecular , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/metabolismo , Sistemas de Lectura Abierta/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Uracil-ADN GlicosidasaRESUMEN
We sequenced the region of the bovine herpesvirus type 1 (BHV-1) genome corresponding to map units 0.172-0.230 (7964 bp), representing the UL39, UL38, and UL37 homologues of herpes simplex virus which encode the large subunit of ribonucleotide reductase (RR) and components of the viral capsid and the tegument, respectively. To discriminate between two potential initiator AUGs of the UL39 gene, the 5' end of the mRNA was mapped by S1 nuclease protection assays. Comparison of the amino acid sequences of the three BHV-1 proteins with analogous polypeptides from several other herpesviruses revealed significant levels of homology. We also compared the expression kinetics of the large (R1, UL39) versus the small (R2, UL40) RR subunits during the course of in vitro BHV-1 infection by Western blotting using specifically developed and calibrated antisera. Our results show that the R1 protein was synthesized earlier than its R2 counterpart. Moreover, the R1 protein accumulated to a higher level than the R2 protein even though the R2 transcript was in greater abundance than the R1 mRNA. This is discussed with regard to the translational efficiency of their transcripts.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Bovino 1/genética , Ribonucleótido Reductasas/genética , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Secuencia de Bases , Cápside/genética , Bovinos , Codón Iniciador/genética , Genes Virales/genética , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/inmunología , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Mensajero/análisis , ARN Viral/análisis , Ribonucleótido Reductasas/biosíntesis , Ribonucleótido Reductasas/inmunología , Homología de Secuencia de Aminoácido , Proteínas Estructurales Virales/genéticaRESUMEN
Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.
Asunto(s)
Herpesvirus Bovino 1/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Timidina Quinasa/genética , Animales , Secuencia de Bases , Southern Blotting/veterinaria , Bovinos , Células Cultivadas , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5'-iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.
Asunto(s)
Deleción Cromosómica , Herpesvirus Bovino 1/inmunología , Rinotraqueítis Infecciosa Bovina/prevención & control , Timidina Quinasa/genética , Vacunas Virales , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Bovinos , Sondas de ADN/química , ADN Viral/química , Femenino , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Vacunas Virales/efectos adversos , Vacunas Virales/inmunologíaRESUMEN
The gene encoding bovine herpesvirus 1 (BHV 1) deoxyuridine triphosphatase (dUTPase) was isolated by a PCR procedure using degenerate oligonucleotide primers whose sequences were based upon conserved motifs commonly present in dUTPase genes. This gene was found to reside between 0.059 and 0.066 map units in the BHV 1 Cooper strain. DNA sequence analysis of this region revealed an open reading frame of 975 base pairs capable of encoding 325 amino acids. The deduced amino acid sequence of the open reading frame exhibits significant homology with dUTPases of other herpesviruses (including human herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus), and it contains five conserved amino acid motifs characteristics of all dUTPases identified to date. A mutant virus carrying a partial deletion of the putative dUTPase gene was made and was found to lack virus-encoded dUTPase activity. This further confirmed that we have identified the BHV 1 dUTPase gene. In addition, a further analysis of the genomic fragment which contains the dUTPase coding sequence revealed an additional 288-base-pair open reading frame which appears to be colinear with the HSV 1 UL49.5 gene. The deduced amino acid sequence of this open reading frame is significantly homologous to the HSV 1 UL49.5 gene product, and as with UL49.5, it contains a potential signal sequence and transmembrane domain characteristic of membrane-associated proteins. These results suggest that this open reading frame represents the BHV 1 homolog of the HSV 1 UL49.5 gene. Since our dUTPase negative mutant was fully viable and since the mutant was constructed such that the UL49.5 gene was also deleted, both the dUTPase and the UL49.5 gene homolog are not required for virus growth in cell culture.
Asunto(s)
Herpesvirus Bovino 1/genética , Monoéster Fosfórico Hidrolasas/genética , Pirofosfatasas , Simplexvirus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Clonación Molecular , ADN Viral , Herpesvirus Bovino 1/enzimología , Datos de Secuencia Molecular , Mutagénesis , Monoéster Fosfórico Hidrolasas/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Simplexvirus/enzimologíaRESUMEN
The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.
Asunto(s)
Genes Virales/genética , Herpesvirus Bovino 1/enzimología , Ribonucleótido Reductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Línea Celular , Clonación Molecular , Herpesvirus Bovino 1/genética , Datos de Secuencia Molecular , Mapeo Restrictivo , Ribonucleótido Reductasas/química , Transcripción Genética/genéticaRESUMEN
The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase-negative (TK-) deletion mutant virus. Twelve heifers were inoculated IV at 25 to 29 weeks of pregnancy with either TK- or thymidine kinase-positive (TK+) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of TK+ inoculates were slightly higher and remained above normal a few days longer than in TK- inoculates. Viremia was detected in 5 of 6 TK+ inoculates and in all 6 TK- inoculates. More virus isolations were made from nasal and vaginal swab specimens of TK+ inoculates than from swab specimens of TK- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the TK- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given TK+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the TK gene reduces abortifacient activity of bovine herpesvirus-1.
Asunto(s)
Aborto Veterinario/microbiología , Enfermedades de los Bovinos/microbiología , Herpesvirus Bovino 1/patogenicidad , Timidina Quinasa/genética , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/análisis , Autorradiografía , Temperatura Corporal , Bovinos , Femenino , Herpesvirus Bovino 1/enzimología , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/inmunología , Inmunohistoquímica , Mutagénesis , Mucosa Nasal/microbiología , Pruebas de Neutralización , Sistemas de Lectura Abierta , Placenta/microbiología , Plásmidos , Embarazo , Mapeo Restrictivo , Vagina/microbiología , Viremia/veterinariaRESUMEN
The nucleotide and predicted amino acid sequence of the thymidine kinase (TK) gene of N 569, a bovine encephalitis herpesvirus (BEHV), has been determined and compared with those of avian, bovine and other mammalian herpesvirus TK genes. Striking differences were observed between the nucleotide sequence of this BEHV TK gene and those reported for bovine herpesvirus 1 (BHV-1). A total of 118 base changes, 39 base deletions and 14 base insertions were identified relative to the TK sequence of a BHV-1.2a strain, resulting in a net loss of seven residues. Comparison of the TK sequences of BEHV and the BHV-1 Q 3932 strain with that reported for the BHV-1 6660 strain suggested that the latter may have contained sequencing errors. The most significant difference between the BEHV TK sequence and those of BHV-1 strains was the absence of a contiguous stretch of ten codons.
Asunto(s)
ADN Viral , Herpesvirus Bovino 1/genética , Timidina Quinasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Herpesvirus Bovino 1/enzimología , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
On the basis of their restriction endonuclease digestion patterns, four Australian bovine herpesvirus type 1 (BHV-1) isolates were classified as belonging to the BHV-1.2a subtypes. The thymidine kinase (TK) genes of all four BHV-1.2a isolates were located on a 3.5 kb SalI restriction fragment. This is in contrast to North American and European BHV-1.1 isolates whose TK genes are contained on a 2.6 to 2.8 kb SalI fragment. The restriction fragments containing the TK genes were cloned into phagemid vectors and their sequences determined using the dideoxynucleotide chain termination method. The BHV-1.2a isolates possessed identical TK gene sequences, which differed from previously published TK sequences for the LA and 6660 BHV-1.1 strains. In addition to five single base alterations, there were six separate base insertions which resulted in two major frameshifts which spanned an area of 72 amino acids or 20% of the expressed TK gene product. The predicted amino acid sequence exhibited a higher degree of similarity to other herpesvirus TKs, suggesting that previously published TK gene sequences may have been incorrect. The present nucleotide sequence and corresponding amino acid composition reinforces previous observations concerning regions of herpesvirus TK amino acid conservation and should assist in future studies into the evolution and functional domains of herpesvirus TKs.
Asunto(s)
Genes Virales , Herpesvirus Bovino 1/genética , Timidina Quinasa/genética , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Australia , Secuencia de Bases , ADN Viral/genética , Variación Genética , Herpesvirus Bovino 1/clasificación , Herpesvirus Bovino 1/enzimología , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
Five thymidine kinase (TK)-deficient mutants (B1 to B5) of bovine herpesvirus type 1 (BHV-1) were isolated by selection for resistance to the nucleotide analogue bromovinyldeoxyuridine. The genetic lesion in mutant B1 was localized in a 2.7 kb SalI-SalI subfragment (fTK2.7) which maps between 0.456 and 0.475 within the HindIII A fragment of the BHV-1 genome. The tk genes from wild-type and the TK-mutants B1 to B5 were cloned and sequenced using eight unique synthetic primers designed from a published sequence. The BHV-1 tk gene sequence for the strain 6660 contained some differences compared with that published previously for strain LA. Alignment of the predicted amino acid sequence of the BHV-1 TK polypeptide with different herpesvirus TKs revealed five strongly conserved regions and also identified putative functional relationships with other enzymes. Several interesting features were apparent in the tk gene sequences from the TK- mutants. The TK mutant B1 was a typical frameshift and chain termination mutant due to the deletion of a single base. The tk gene sequence of mutant B2 revealed the deletion of three bases resulting in the loss of valine at amino acid residue 174 of the TK polypeptide. The tk genes of mutants B3 to B5 contained an identical change of a single base addition resulting in frameshift and premature chain termination. In contrast to wild-type BHV-1, the TK-defective mutants were incapable of adsorbing TK-neutralizing antibodies from serum.
Asunto(s)
ADN Viral/genética , Herpesvirus Bovino 1/genética , Timidina Quinasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Herpesvirus Bovino 1/enzimología , Datos de Secuencia Molecular , Mutación , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Similar open reading frames (ORF) overlapping the thymidine kinase (TK) gene on the opposite strand or being close to it have been identified in 8 herpesviruses (Jacobson et al., 1989). Study of the DNA sequence of BHV-1 TK gene published by Mittal and Field (1989) and Kit and Kit (1986) revealed the existence of a similar ORF in this virus genome, too.
Asunto(s)
ADN Viral/genética , Herpesvirus Bovino 1/genética , Timidina Quinasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Herpesvirus Bovino 1/enzimología , Datos de Secuencia MolecularRESUMEN
The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334-0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Herpesvirus Bovino 1/genética , Secuencia de Aminoácidos , Enzimas de Restricción del ADN , Genes Virales , Herpesvirus Bovino 1/enzimología , Datos de Secuencia Molecular , Hibridación de Ácido NucleicoRESUMEN
Bovine herpesvirus 1 has been reported to contain a thymidine kinase (tk) gene which is nonessential for virus replication. We have isolated a thymidine kinase-negative mutant of the virus and localized the mutation by marker rescue experiments to a 1.1-kilobase BglII-SalI fragment which maps at 0.47 to 0.48 on the bovine herpesvirus 1 genomic map. A thymidine kinase-negative bovine cell line isolated in our laboratory was used in these studies.
Asunto(s)
Genes Virales , Herpesvirus Bovino 1/genética , Timidina Quinasa/genética , Animales , Bovinos , Línea Celular , Clonación Molecular , Enzimas de Restricción del ADN , ADN Viral/genética , Desoxirribonucleasa HindIII , Marcadores Genéticos , Herpesvirus Bovino 1/enzimología , Mutación , FenotipoRESUMEN
To test the safety and efficacy of a thymidine kinase-negative (TK-), temperature-resistant (TR) mutant of bovine herpes virus-1 (BHV-1) in pregnant cows, seronegative cows, 2-5 months pregnant, were vaccinated intramuscularly (i.m.) or intravaginally (i.vag.) with this candidate vaccine virus. I.m. vaccinated cows did not shed virus i.vag. or intranasally (i.n.), but i.vag. vaccinated cows replicated virus i.vag. for 8-9 days postvaccination (p.v.) Some of the cows were challenge exposed i.n. at 46 days p.v. with virulent TK+ BHV-1(Cooper). Vaccinated cows showed no clinical disease signs p.v. or postchallenge and responded anamnestically postchallenge. All cows delivered live calves. Pre-colostrum sera of the calves were negative for BHV-1 antibodies.